Prevention of pertussis using adenylate cyclase deficient Bordetella strains

ABSTRACT

A CyaA-deficient  B. pertussis  mutant was constructed and used in a vaccine. The  pertussis -specific antibody profile and Th17 response induced by vaccination with the mutant was surprisingly comparable to that induced by  B. pertussis  strains not deficient in CyaA.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 14/870,487 filed on Sep. 30, 2015, which claims thepriority of U.S. provisional patent application Ser. No. 62/058,172filed on Oct. 1, 2014.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 28, 2015, isnamed 7056-0062_SL.txt and is 1,212 bytes in size.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Not applicable.

FIELD OF THE INVENTION

The invention relates generally to the fields of microbiology,immunology, vaccinology, sero-epidemiology, biochemistry and medicine.More particularly, the invention relates to mutated strains Bordetellapertussis having deficient adenylate cyclase (CyaA) and their use asvaccines.

BACKGROUND

Despite high vaccination coverage, Bordetella pertussis infectionremains endemic and reports of increasing incidence in Australia,Canada, and Europe have been accumulating for the past twenty years.2012 was the year with the highest whooping cough incidence in US andcomparable outbreaks occurred also in the UK and Netherlands, inproportion of inhabitant numbers. Adaptation of the circulatingpertussis strains to the vaccines, as well as the waning and/orsuboptimal efficacy of vaccine-mediated immunity during adolescence hasbeen proposed to account for resurgence, with infected adolescent andadult populations being the major transmitters of disease in communityrepresenting a potential reservoir for disease transmission to youngchildren who are yet to be fully vaccinated. Furthermore, with thechanging epidemiology, pertussis is increasingly becoming a real burdenalso in adults that experience long-lasting and very heavy cough periodsof duration in weeks to months. This underscores the need to pursueresearch efforts on this disease in order to provide suitable protectionto the most vulnerable populations.

One approach to developing new vaccines to prevent B. pertussisinfection is to use live but attenuated Bordetella bacteria as anantigenic agent. Creating such vaccines remains a challenging endeavorbecause, in order to be safe and effective, the attenuation must removetoxicity while still preserving sufficient antigenicity and viability.Deletion or mutation of various B. pertussis components can be lethal tothe bacteria, can render the bacteria unable to colonize a subject,and/or unable to induce a sufficiently protective immune response.

Previously, after much work and numerous failures, a highly attenuatedB. pertussis strain named BPZE1 [deposited with the Collection Nationalede Cultures de Microorganismes (CNCM, Institut Pasteur, 25 Rue duDocteur Roux, F-75724, Paris, Cedex 15, France) on Mar. 9, 2006 underthe number CNCM I-3585] was developed. This strain producesenzymatically inactive pertussis toxin (PTX), no dermonecrotic toxin(DNT), and only trace amounts of tracheal cytotoxin (TCT). It was alsoshown to be genetically stable and safe in preclinical models and a 12subject clinical trial. Given that B. pertussis virulence factors haveevolved to promote colonization and prevent infection clearance (e.g.,PTX inhibits neutrophils/macrophage recruitment and TCT inducesciliostasis), BPZE1's ability to colonize the lung was impaired.Nonetheless, it was surprisingly found that intranasally (i.n.)administered BPZE1 was able to colonize the lungs of mice sufficientlyto induce a strong protective humoral and cellular immunity.

SUMMARY

Described herein is the development of a new live, attenuated B.pertussis strain that is useful for inducing protective immune responsesagainst B. pertussis infection. The newly developed strain is deficientin adenylate cyclase toxin (CyaA) as well as PTX, DNT, and TCT. CyaA isa polypeptide that is synthesized as an inactive protoxin which, afterposttranslational fatty acylation, is converted to an active toxin. TheC-terminal domain of the active toxin binds to target cell membranesallowing the N-terminal catalytic domain to enter the target cell'scytosol where it is activated by Ca2+/calmodulin to catalyze theconversion of cellular ATP into cAMP. Increased cAMP interferes with thesignaling pathways in immune cells (including macrophages andneutrophils) and reduces phagocytic activity. CyaA has been previouslyreported to be an important colonization factor for B. pertussis thathelps initiate infection. Because the newly developed strain isdeficient in two key phagocyte-neutralizing factors, CyaA and PTX, andBPZE is already impaired in its ability to colonize, it was surprisingto discover that this quadruple mutant was also able to sufficientlycolonize the lungs of subjects to induce protective immunity againstlater challenges with wild-type B. pertussis.

Accordingly, in one aspect, the invention features a live attenuatedBordetella strain which has been engineered to reduce or remove CyaAactivity while retaining the ability to colonize the lungs of a subjectand induce protective immunity against later challenges with wild-typeB. pertussis. Preferably the live attenuated Bordetella strain also hasbeen engineered to reduce or remove the activity of at least one (e.g.,1, 2, or 3) of: PTX, DNT, and TCT.

It another aspect, the present invention includes a method of using alive attenuated Bordetella strain which has been engineered to reduce orremove CyaA activity and at least one (1, 2, or 3) of PTX activity, DNTactivity, and TCT activity to elicit an immune response which protects amammalian subject (e.g., a human being) against B. pertussis infection.

In the various aspects of the invention, the live attenuated Bordetellastrain which has been engineered to reduce or remove CyaA, PTX, DNT, andTCT activity is BPAL10 [deposited in accordance with the requirements ofthe Budapest Treaty with the National Measurement Institute (“NMI”),1/153 Bertie Street, Port Melbourne, Victoria, Australia 3207 on Oct.23, 2015 under accession number V15/032164.

Also within the invention is a vaccine including a pharmaceuticallyacceptable carrier and a live attenuated Bordetella strain that has beenrendered deficient in functional CyaA, PTX, dermonecrotic toxin (DNT),and tracheal cytotoxin (TCT), but retains the ability to colonize amammalian subject's lungs and induce a protective immune responseagainst Bordetella infection. The strain can include a gene encodingCyaA which has been mutated such that the strain fails to produce CyaAand/or a gene encoding PTX which has been mutated such that the strainfails to produce PTX. The strain can also lack a gene encodingfunctional DNT and/or a functional wild-type ampG gene (which can bereplaced by a heterologous ampG gene).

Further within the invention is a method of protecting a mammaliansubject from developing pertussis. This method includes the step ofadministering to the mammalian subject a vaccine including a sufficientamount of one or more of the live attenuated Bordetella strainsdescribed above to elicit immune response that prevents the mammaliansubject from developing pertussis after exposure to B. pertussis.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. Commonly understood definitions ofbiological terms can be found in Rieger et al., Glossary of Genetics:Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991;and Lewin, Genes V, Oxford University Press: New York, 1994.

As used herein, the abbreviation “CyaA” refers to adenylate cyclasetoxin, which is a virulence factor synthesized by Bordetellae. CyaA is apolypeptide that is synthesized as an inactive protoxin which, afterposttranslational fatty acylation, is converted to an active toxin. TheC-terminal domain of the active toxin binds to target cell membranesallowing the N-terminal catalytic domain to enter the target cell'scytosol where it is activated by Ca2+/calmodulin to catalyze theconversion of cellular ATP into cAMP. Increased cAMP interferes with thesignaling pathways in immune cells (including macrophages andneutrophils) and reduces reduce their phagocytic activity.

As used herein, the abbreviation “PTX” refers to pertussis toxin, whichsynthesizes and secretes an ADP-ribosylating toxin. PTX is composed ofpolypeptides S1 to S5, the enzymatically active moiety is called S1. PTXhas a 34 amino acid signal sequence, while the mature chain consists ofamino acids 35 to 269. PTX is the major virulence factor expressed by B.pertussis. The A moiety of these toxins exhibit ADP-ribosyltransferaseactivity and the B portion mediates binding of the toxin to host cellreceptors and the translocation of A to its site of action.

As used herein the abbreviation “DNT” refers to pertussis dermonecrotictoxin, which is a heat labile toxin that induces localized lesions inmice and other laboratory animals when it is injected intradermally. Itis lethal to mice when it is injected in low doses intravenously, and isconsidered to be a virulence factor for the production of turbinateatrophy in porcine atrophic rhinitis.

As used herein the abbreviation “TCT” refers to tracheal cytotoxin,which is a virulence factor synthesized by Bordetellae. TCT is apeptidoglycan fragment and has the ability to induce interleukin-1production and nitric oxide synthase. It has the ability to cause stasisof cilia and has lethal effects on respiratory epithelial cells.

The term “functional” when referring to a toxin means that the toxinretains wild-type activity. For example, a Bordetella strain that hasbeen rendered deficient in functional CyaA, PTX, DNT, or TCT exhibitsless than 50% (e.g., less than 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1%) ofat least one of that toxin's native activity described in the precedingfour paragraphs.

The term “mammal”, “mammalian subject” or “subject” encompasses any ofvarious warm-blooded vertebrate animals of the class Mammalia, includinghuman beings, characterized by a covering of hair on the skin and, inthe female, milk-producing mammary glands for nourishing the young.

The term “attenuated” means a weakened, less virulent Bordetella strainthat is capable of stimulating an immune response and creatingprotective immunity, but does not cause significant illness.

The term “Bordetella strain” encompasses strains from Bordetellapertussis, Bordetella parapertussis and Bordetella bronchiseptica.

The expression “Bordetella infection” means an infection caused by atleast one of the three following strains: Bordetella pertussis,Bordetella parapertussis and Bordetella bronchiseptica.

By “child” is meant a person or a mammal between 6 months and 12 yearsof age.

By the term “newborn” is meant, a person or a mammal that is between 1day old and 24 weeks of age.

The term “treatment” as used herein is not restricted to curing adisease and removing its causes but particularly covered means to cure,alleviate, remove or lessen the symptoms associated with the disease ofinterest, or prevent or reduce the possibility of contracting anydisorder or malfunction of the host body.

The terms “protection” and “prevention” are used herein interchangeablyand mean that an infection by Bordetella is impeded.

“Prophylaxis” means that to prevent or reduce the pathological effectsor symptoms of an infection.

The term “immunogenic composition” means that the composition can inducean immune response and is therefore antigenic. By “immune response”means any reaction by the immune system. These reactions include thealteration in the activity of an organism immune system in response toan antigen and may involve, for example, antibody production, inductionof cell-mediated immunity, complement activation or development ofimmunological tolerance.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and material are described below. All publications andpatent applications mentioned herein are incorporated by reference intheir entirety. In the case of conflict, the present specification,including definitions will control. In addition, the particularembodiments discussed below are illustrative only and not intended to belimiting.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic diagram of the probe design for Southern blotanalysis of BPAL10. Restriction enzymes, SacI and SphI are indicated byarrows.

FIG. 1B is a Southern blot analysis of the CyaA locus in BPZE1 andBPAL10.

FIG. 1C is a Western blot analysis of CyaA, FHA and PTX production inwhole cell extracts and supernatants from BPZE1 and BPAL10.

FIG. 1D is a photograph showing hemolytic activity of BPZE1 and BPAL10on blood agar observed upon incubation at 37° C. for 3 days.

FIG. 2 is a series of graphs showing the phenotypic characterization ofBPAL10. (A) In vitro growth kinetic of BPAL10 (open circle) and BPZE1(solid circle) in SS liquid culture. Exponential liquid bacterialpre-cultures in SS medium were used to inoculate a fresh culture mediumat an initial OD600 nm of 0.5. Growth of bacteria was monitored overtime at the indicated time points. (B) In vitro cell invasion assay ofBPAL10 (open circle) and BPZE1 (solid circle) in J774.A1 cells. Theinfected cells (MOI 20) were incubated for 1 h at 37° C. and 5% CO₂,washed and further incubated with gentamycin for 2 h to removeextracellular bacteria. Cells were washed, lysed and appropriatedilutions were plated for colony counting. P values of <0.01 (**)compared to BPZE1-immunized mice were considered significant. Eachsample was performed in quadruplicate. The experiment was repeated twiceindependently. (C) In vitro adherence of BPAL10 (open bar) and BPZE1(black bar) to murine macrophage-like J774.A1 cells and human pulmonaryepithelial A549 cells as indicated. 2×10⁵ mammalian cells per well wereinfected with BPAL10 or BPZE1 at MOI 20, incubated for 1 h at 4° C.,washed, lysed, and appropriate dilutions were plated for colonycounting. Each sample was performed in quadruplicate. The experiment wasrepeated twice independently. (D) Lung colonization profile of BPAL10 in3 week-old BALB/c mice. Three week-old BALB/c mice were administeredintranasally with either 5×10⁵ CFU or 5×10³ CFU of BPAL10 or BPZE1 asindicated. At the indicated time points, 4 mice per group wereeuthanized and their lungs were processed for colony counting. Theresults are expressed as means (±standard error) CFUs from 4 mice pergroup per time point, and are representative of two independentexperiments. P values <0.01 (**) compared to BPZE1-immunized mice wereconsidered significant. The dashed line represents the detection limitof the number of CFU present in the lungs.

FIG. 3 is a series of graphs showing B. pertussis-specific antibodyresponses in BPAL 10-immunized infant BALB/c mice. Three week-old BALB/cmice were intranasally administered with 5×10³ CFU of BPAL10 or BPZE1 asindicated. Bronchoalveolar lavage fluids (BALFs) (A and B) and sera (Cand D) were collected 2 months post-infection and total IgA (A and C)and IgG (B and D) antibody responses to B. pertussis whole cell lysatewere determined by ELISA on individual sera diluted 1/50 and neat BALFs.P values of <0.01 (**) compared to BPZE1-immunized mice were consideredsignificant.

FIG. 4 is a series of graphs and histograms showing cytokine expressionprofile of BPAL10-primed splenocytes. Three week-old BALB/c mice wereintranasally administered with 5×10³ CFU of BPAL10 or BPZE1 and theirspleens were harvested 2 months post-infection for in vitrore-stimulation with whole cell B. pertussis lysate. After 60 h ofculture, levels of IFN-γ (A), IL-4 (B) and IL-17 (C) produced in theculture supernatants were quantified by ELISA. The data are expressed asthe means±standard errors of the means (SEM) of duplicates andrepresentative of two independent experiments. P values of <0.01 (**)compared to BPZE1-immunized mice were considered significant. (D)Re-stimulated splenocytes were labeled for surface expression of CD4,and stained for intracellular IFNγ expression. The percentages of doublepositive CD4+ IFNγ+ cells were determined by flow cytometry. Results arerepresentative of three independent experiments.

FIG. 5 is a series of graphs showing challenge experiments with virulentB. pertussis. Three week-old BALB/c mice were intranasally immunizedwith PBS (naive) (black bar), BPZE1 (openbar) or BPAL10 (gray bar) usingtwo doses, 5×10⁵ CFU (A) or 5×10³ CFU (B), and challenged with 5×10⁶ CFUof virulent B. pertussis BPSM one month post-immunization. At theindicated time points, 4 animals per group were euthanized and theirlungs were harvested and processed for colony counting. The results areexpressed as the mean of CFU from 4 mice (+/−standard deviations). Thedashed line represents the detection limit of the number of CFU presentin the lungs. P value of <0.01 (**) compared to BPZE1 mice wereconsidered significant.

DETAILED DESCRIPTION

Described herein are new Bordetella strains and methods of using suchstrains to generate protective immune responses against infectiousagents. The present invention is not limited to only the mutantsdescribed above. Other additional mutations can be undertaken such aslipopolysaccharide (LPS) deficient mutants, filamentous hemagglutinin(FHA), and any of the bvg-regulated components.

General Methods

Methods involving conventional microbiological, immunological, molecularbiological, and medical techniques are described herein. Microbiologicalmethods are described in Methods for General and Molecular Microbiology(3d Ed), Reddy et al., ed., ASM Press. Immunological methods aregenerally known in the art and described in methodology treatises suchas Current Protocols in Immunology, Coligan et al., ed., John Wiley &Sons, New York. Techniques of molecular biology are described in detailin treatises such as Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, Sambrook et al., ed., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 2001; and Current Protocols in MolecularBiology, Ausubel et al., ed., Greene Publishing and Wiley-Interscience,New York. General methods of medical treatment are described in McPheeand Papadakis, Current Medical Diagnosis and Treatment 2010, 49thEdition, McGraw-Hill Medical, 2010; and Fauci et al., Harrison'sPrinciples of Internal Medicine, 17th Edition, McGraw-Hill Professional,2008.

Bordetella Strains with Reduced or Removed CyaA and PTX Activity

The vaccines and methods described herein are based on live attenuatedBordetella strains that have been rendered deficient in functionalvirulence factors including CyaA and PTX, but retain the ability tocolonize a mammalian subject's lungs and induce a protective immuneresponse against Bordetella infection. Preferably such strains have alsobeen rendered deficient in functional DNT and/or TCT. The strains caninclude a gene encoding CyaA which has been mutated such that the strainfails to produce functional CyaA and/or a gene encoding PTX which hasbeen mutated such that the strain fails to produce functional PTX. Thestrains can also lack a gene encoding functional DNT and/or a functionalwild-type ampG gene (which can be replaced by a heterologous ampG gene).

The live attenuated Bordetella strains described above can be made bymethods known in the art such as those described in the Examples sectionbelow. The starting strain can be any suitable Bordetella species.Examples of Bordetella species include B. pertussis, B. parapertussis,and B. bronchiseptica. B. pertussis is preferred for use as the startingstrain for vaccines and methods for preventing pertussis infection. Anumber of suitable Bordetella strains for use as starting strains areavailable from established culture collections (e.g., the American TypeCulture Collection in Manassas, Va.) or can be isolated from naturalreservoirs (e.g., a patient having pertussis) by known techniques (e.g.,Aoyama et al., Dev. Biol. Stand, 73:185-92, 1991).

A variety of methods are known in the art for attenuating an infectiousbacterial strain. These include passaging the strain in vitro untilvirulence is lost, non-specific chemical mutagenesis followed byscreening and selection based on phenotype, and using targeted molecularbiology techniques such as those described in the Examples section below(including allelic exchange) and in Methods for General and MolecularMicrobiology (3d Ed), Reddy et al., ed., ASM Press. Using these methods,the genes encoding CyaA, PTX, and/or DNT can be deleted or mutated to anenzymatically inactive form (which is preferred where it is desired toretain the toxin's antigenicity). TCT production can be significantly(e.g., > than 99.99, 99.90, 99.8, 99.7, 99.6, 99.5, 99.0, 98, 97, 96,95, or 90%) reduced by replacing the native ampG gene (unlike otherspecies, B. pertussis ampG does not actively recycle TCT-containingpeptidoglycan) with a heterologous (e.g., from E. coli or another gramnegative species) ampG gene, or by mutating the native ampG gene suchthat it is active at recycling peptidoglycan.

Modification of a starting strain to reduce or remove toxin activity canbe confirmed by sequencing the genomic DNA or genes encoding the toxinsof the modified strains. Southern, Northern, and/or Western blottingmight also be used to confirm that the target genes have been deleted orthat expression of the target proteins has been reduced or removed.Enzyme activity can also be evaluated to confirm reduction or removal oftoxin activity. Once the modifications have been confirmed, the modifiedstrains can be tested for the ability to colonize a subject and toinduce protective immunity against Bordetella infection by known methodssuch as those described in the Examples section below.

Vaccines

The live attenuated Bordetella strains described herein can be used invaccines that protect a mammalian subject from developing a Bordetellainfection (e.g., pertussis) or at least reduce the symptoms of such aninfection. For use in a vaccine, the live attenuated Bordetella strainsare formulated with a pharmaceutically acceptable excipient. Examples ofpharmaceutically acceptable excipients include, e.g., buffered salinesolutions, distilled water, emulsions such as an oil/water emulsion,various types of wetting agents sterile solutions, and the like.

The vaccines can be packaged in unit dosage form for convenientadministration to a subject. For example, a single dose of between 1×10⁴to 1×10⁷ (e.g., 1×10⁴, 5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, or 1×10⁷+/−10,20, 30, 40, 50, 60, 70, 80, or 90%) live bacteria of the selectedattenuated Bordetella strain and any excipient can be separatelycontained in packaging or in an administration device. The vaccine canbe contained within an administration device such as a syringe, sprayingdevice, or insufflator.

Methods of Eliciting Immune Responses to Protect Against Pertussis

The vaccines described herein can be administered to a mammalian subject(e.g., a human being, a human child or neonate, a human adult, a humanbeing at high risk from developing complications from pertussis, a humanbeing with lung disease, and a human being that is or will becomeimmunosuppressed) by any suitable method that deposits the bacteriawithin the vaccine in the respiratory tract. For example, the vaccinesmay be administered by inhalation or intranasal introduction, e.g.,using an inhaler, a syringe, an insufflator, a spraying device, etc.While administration of a single dose of between 1×10⁴ to 1×10⁷ (e.g.,1×10⁴, 5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, or 1×10⁷+/−10, 20, 30, 40, 50,60, 70, 80, or 90%) live bacteria is typically sufficient to induceprotective immunity against developing a Bordetella infection such aspertussis, one or more (1, 2, 3, 4, or more) additional doses might beadministered in intervals of 4 or more days (e.g., 4, 5, 6, or 7 days;or 1, 2 3, 4, 5, 6, 7, or 8 weeks) until a sufficiently protectiveimmune response has developed. The development of a protective immuneresponse can be evaluated by methods known in the art such asquantifying Bordetella-specific antibody titers and measuring ofBordetella antigen-specific T cells responses (e.g., using an ELISPOTassay). In cases were a vaccine-induced protective immune response haswaned (e.g., after 1, 2, 3, 4, 5, 10 or more years from the lastvaccination) a subject may again be administered the vaccine in order toboost the anti-Bordetella immune response.

EXAMPLES Methods

Bacterial growth conditions: BPSM is a streptomycin-resistant TohamaI-derived B. pertussis strain. BPAL10 was derived from B. pertussisBPZE1, a BPSM derivative strain producing inactivated pertussis toxin(PTX), no dermonecrotic toxin (DNT) and background levels of trachealcytotoxin (TCT). B. pertussis strains were cultivated at 37° C. for 72 hon Bordet-Gengou (BG) agar (Difco, Detroit, Mich.) supplemented with 1%glycerol, 10% defibrinated sheep blood, and 100 μg/ml streptomycin(Sigma Chemical CO., St Louis, Mo.). Liquid cultures were performed inStainer-Scholte (SS) medium containing 1 g/L heptakis(2,6-di-o-methyl)b-cyclodextrin (Sigma).

Cell lines: The human pulmonary epithelial cell line A549 (ATCC CCL-185)and the mouse macrophage cell line J774A.1 (ATCC TIB-67) were culturedaccording to the ATCC guidelines.

Construction of the CyaA-deficient BPZE1 strain: The CyaA-deficientBPZE1 strain, named BPAL10 was obtained by a double homologousrecombination strategy. A 999-bp PCR1 DNA fragment encompassing the989-bp sequence upstream the first nucleotide of the cyaA ORF and thefirst 10-bp of cyaA was first PCR amplified from purified BPZE1chromosomal DNA using the primers, 5′-TTTCTAGAGGCGGTGCCCCGGCCTCG-3′(XbaI site underlined) (SEQ ID NO:1) and5′-TTGAGCTCTCGCACCGACGCAACCGGTG-3′ (SEQ ID NO:2). Similarly, a 996-bpPCR2 fragment including the 986-bp sequence downstream the STOP codon ofcyaA ORF and the last 10-bp of cyaA, was PCR-amplified using the primers5′-TTGAGCTCAGCGCCGTGAATCACGGCCC-3′ (SEQ ID NO:3) and5′TTAAGCTTAGCAAGGCAACGCCGCCAGC-3′ (HindIII site underlined) (SEQ IDNO:4). Both PCR1 and PCR2 fragments were sequentially cloned intoplasmid pJQmp200rpsL18 to obtain the pJQ-PCR2-PCR1 construct using XbaIand HindIII restriction sites. BPZE1 bacteria were electroporated withpJQ-PCR2-PCR1 for integration via double homologous recombination at thecyaA locus to obtain the recombinant B. pertussis BPAL10.

Southern blot: Chromosomal DNA was extracted and purified from BPZE1 andBPAL10 bacteria using Genomic-tip 100/G Anion-Exchange Resin (Qiagen)and Genomic DNA Buffer Set (Qiagen) according to the manufacturer'sinstructions. Chromosomal DNA (1 μg) from BPZE1 and BPAL10 was digestedwith restriction enzymes for 4 h and subjected to 1.5% agarose gelelectrophoresis. The agarose gel containing the digested DNA waschemically treated and transferred onto a nitrocellulose membrane(Millipore) according to Roche's DIG application manual. The membranewas UV-fixed for 1 min and equilibrated with 10 ml pre-heated DIG EasyHyb solution (Roche) at 65° C. for 20 min, with gentle agitation. Adigoxigenin (DIG)-labeled probe was amplified using the PCR DIG ProbeSynthesis Kit (Roche) according to the manufacturer's instructions. Forhybridization, about 5-25 ng/ml of heat-denatured DIG-labeled DNA probein DIG Easy Hyb solution was incubated with the membrane overnight at65° C. Detection was performed using alkaline phosphatase(AP)—conjugated anti-DIG antibody (Roche) at a dilution of 1:5000. Themembrane was developed using NBT/BCIP AP substrate (Chemicon).

Western blot: Mid- to late exponentially grown bacteria in SS medium (10ml) were centrifuged at 7000 rpm for 15 min at room temperature. Thesupernatant was concentrated 10 times using the 30 kDa cut-off Ultra-4Centrifugal Filter Device (Amicon) following the manufacturer'sprotocol. The bacterial pellet was re-suspended in 500 μl of ultrapurewater. An equal volume of 2× loading buffer [0.125 M TriseHCl (pH 6.8),10% (w/v) SDS, 10% (v/v) glycerol, 10% (v/v) 2-mercaptoethanol and 1drop of Bromophenol blue] was added before heating at 95° C. for 10 min.Chromosomal DNA was sheared by passing the suspension 10 times through a27G needle followed by heating at 95° C. for 15 min prior to analysis bySDS-PAGE using 8% polyacrylamide gels. The proteins wereelectro-transferred onto nitrocellulose membranes and incubated withmouse anti-CyaA 9D4 monoclonal antibodies (Santa Cruz Inc., US) dilutedat 1:1000, anti-FHA monoclonal anti-bodies (National Institute forBiological Standards and Control, UK) diluted at 1:40,000 and anti-PTXmonoclonal antibodies (National Institute for Biological Standards andControl, UK) diluted at 1:4000 in Tris-buffered saline containing 0.1%Tween 20 and 2% bovine serum albumin (BSA). Alkaline phosphatase(AP)-conjugated goat anti-mouse IgG secondary antibodies (Sigma) dilutedat 1:3000 was used for chromogenic detection of the proteins upon addingthe AP substrate (NBT and BCIP reagents, Sigma).

Growth kinetic assay: For in vitro growth kinetics of B. pertussisstrains, fresh SS liquid medium was inoculated with an exponentiallygrown culture at an initial OD_(600 nm) of 0.5. Absorbance at 600 nm ofthe cultures was monitored over time at the indicated time points.

Mammalian cell infection: Bacteria grown on BG plates for 3 days at 37°C. were scraped, washed once in sterile PBS and resuspended in RPMI orDMEM medium; 2×10⁵ mammalian cells per well were cultured for 2 days in24-well plate. The bacteria were added to the cells at a multiplicity ofinfection (MOI) of 20. For adherence assays, the cells were incubatedfor 1 h at 4° C., washed thrice with PBS to remove non-adherentbacteria, lysed with H₂O and appropriate dilutions were plated forcolony counting. For invasion assay, the plates were incubated for 1 hat 37° C. and 5% CO₂, washed thrice with PBS and further incubated at37° C., 5% CO₂ with 100 μg/ml gentamycin for 2 h. Cells were washed,lysed and appropriate dilutions were plated for colony counting. Eachsample was performed in quadruplicate. Each experiment was repeatedtwice independently.

Mouse experiments: Specific-pathogen free BALB/c mice were housed inIndividual Ventilated Cages. For colonization studies, 3 week-old BALB/cmice (CARE) were intranasally (i.n.) infected with the different B.pertussis strains at either 5×10⁵ or 5×10³ CFU/mouse. At the indicatedtime points post-infection, four mice per group were euthanized, andtheir lungs were aseptically removed and homogenized in PBS. Serialdilutions from individual lung homogenates were plated onto BG agar andthe total CFU per lung was determined after 4-5 days incubation at 37°C.

For immunization studies, groups (n=6) of 3-week-old BALB/c mice werei.n. infected with 5×10³ CFU in 20 μl of the different B. pertussisstrains. Bronchoalveolar lavage fluids (BALFs) and sera were collectedtwo months post-immunization from euthanized mice. Individual BALFsamples were recovered by injecting 1 ml of sterile PBS into the lungsof sacrificed animals and performing one lavage step. BALFs were thencentrifuged, and the supernatant was removed and stored at −20° C. forantibody detection.

For challenge experiments, BPAL10- or BPZE1-immunized (5×10³ or 5×10⁵CFU/mouse) mice were i.n. challenged one month post-immunization with5×10⁶ CFU of virulent BPSM strain. They were euthanized at the indicatedtime points and their lung homogenates were plated for colony counting.

All the animal experiments were repeated at least twice independently.

Antibody determination: The anti-B. pertussis antibody responses weremeasured by enzyme-linked immunosorbent assay (ELISA) using BPSM totalcell lysate as coating antigens. Flat 96-well microtiter plates (CorningNUNC) were coated overnight at 4° C. with 50 ml of coating buffer (0.1 MNa₂CO₃—NaHCO₃, pH 9.6) containing 5 μg/ml of BPSM total cell lysate. Thewells were washed thrice with wash buffer (0.1% Tween 20 in PBS), beforeblocking with blocking buffer (0.1% Tween 20, 2% BSA in PBS) for 1 h at37° C. The wells were then washed thrice with wash buffer and 50 μl ofmouse serum diluted in blocking buffer or neat bronchoalveolar lavagefluids (BALFs) were added to each well. After incubation at 37° C. for 2h, the plates were washed thrice before adding 50 μl of 1:3000 dilutedhorseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L)(Bio-rad) and 1:2000 diluted HRP-conjugated goat anti-mouse IgA(Chemicon) secondary antibodies. The plates were again incubated at 37°C. for 1 h followed by three washes. The ELISA was developed by adding50 μl of o-phenylenediamine dihydrochloride (OPD) substrate (Sigma) toeach well and incubating at room temperature for 20 min in the dark. Tostop the reaction, 75 μl of 1 M sulfuric acid was added to each well andabsorbance at 490 nm was measured using the Biorad model 680 MicroplateReader and recorded with the Microplate Manager 5.2 software (Biorad).Antibody isotyping was performed as described above with 1:3000 dilutedHRP-conjugated goat anti-mouse Fcγ Subclass 1, 2a, 2b and 3 specific IgG(Jackson ImmunoResearch).

Intracellular IFNγ staining: Single-cell suspensions were prepared fromspleens of naïve, BPZE1-treated and BPAL10-treated mice (5 mice pergroup). The splenocytes were seeded in 96-well round-bottom plates(Nunc) at a density of 2×10⁶ cells/well in 100 μl RPMI complete medium(RPMI 1640 supplemented with 10% FCS, 5×10⁻⁵ M β-mercaptoethanol, 2 mML-glutamine, 10 mM HEPES, 200 U/ml penicillin, 200 μg/ml streptomycin).RPMI complete medium (100 μl) containing 20 μg/ml of BPSM whole-celllysate was added to the splenocytes. For staining of intracellular IFN,Brefeldin A (One ml of BD GolgiPlug, BD Biosciences) was added to thecultures for the last 5 h to prevent secretion of intracellularcytokines. One million cells were labeled with the eFluor-conjugatedanti-CD4 antibody (Clone RM4-5; BD Biosciences) for 30 min at 4° C.Cells were then washed and fixed with BD Cytofix/Cytoperm™ PlusFixation/Permeabilization Kit, according to manufacturer's protocol (BDBiosciences). To label intracellular IFNγ, cells were incubated withallophycocyanin (APC)-conjugated anti-IFNγ antibodies (clone XMG1.2; BDBiosciences) in the presence of saponin for 30 min at 4° C., washed, andanalyzed by use of a Cyan flow cytometer using Summit software (BeckmanCoulter).

Cytokine measurement: The levels of cytokines in the culturesupernatants from re-stimulated splenocytes were measured by usingindividual detection kits (eBioscience, San Diego, Calif.) according tothe manufacturer's instructions. The experiment was repeated three timesindependently.

Statistical analysis: In the figures, bars represent the means±standarddeviations (SD), and averages were compared using a bidirectionalunpaired Student's t test with a 5% significance level with, **P≤0.01.

Results: CyaA deletion in BPZE1 strain and phenotypic characterization:An unmarked deletion of the BPZE1 chromosomal CyaA-encoding gene cyaAwas obtained by allelic exchange and was confirmed by Southern blotanalyses (FIGS. 1A and B). The mutant strain, named BPAL10, did notproduce CyaA, as shown by immunoblot analysis (FIG. 1C) and expressed nohemolytic activity, as evidenced by absence of the clearing zone upongrowth on blood agar (FIG. 1D). The in vitro growth profile of BPAL10was comparable to that of the parental strain BPZE1 with exponentialgrowth phase up to 27 h followed by a typical stationary phase where theapparent decrease in OD₆₀₀ readings is due to FHA-mediated clumping ofthe bacteria. These observations thus indicated that absence of CyaAdoes not impair the overall in vitro bacterial fitness (FIG. 2A).

An invasion assay was performed using the murine macrophages J774.1.Bacteria and macrophages were incubated at 37° C. and the infected cellswere lysed at different time points post-infection for colony counting.The results indicated that intracellular survival of BPAL10 within themacrophages was comparable to that of the parental counterpart BPZE1,with the exception of the last time point (48 h) at which asignificantly lower number of BPAL10 bacteria was recovered compared toBPZE1 (FIG. 2B).

The adherence properties of the CyaA deficient mutant to the humanpulmonary epithelial cells A549 and to the murine macrophages J774A.1were analyzed upon incubation of the bacteria with the mammalian cellsat 4° C. to prevent internalization. No statistical difference in theadherence capability to both cell lines was noticed between BPZE1 andBPAL10 bacteria (FIG. 2C).

Lung colonization profile: Whether BPAL10 is impaired in the ability tocolonize the mouse respiratory tract was assessed. Since young infantsare the ultimate target population of novel pertussis vaccines, thecolonization profile of BPAL10 was monitored in infant (3 weeks old)BALB/c mice. At an administration dose of 5×10⁵ CFU, BPAL10 displayed amild but significant decrease in its ability to persist in the lungs ofinfant mice compared to BPZE1, as evidenced by significantly lower CFUsrecovered at day 7 and 10 post-infection (p.i.) (FIG. 2D). Thisdifference was even more pronounced at a lower administration dose of5×10³ CFU, with a rapid BPAL10 clearance within 7 days p.i., whereasmore than 2 log₁₀ CFUs of BPZE1 were still recovered at day 10 pi. (FIG.2D).

Local and systemic anti-B. pertussis antibody responses: Antibodyresponses against B. pertussis infection play an important role inprotection, and passive immunization with B. pertussis-specificanti-sera reduces disease severity in infected individuals. Furthermore,mucosal IgA in the respiratory tract can inhibit adherence of B.pertussis to human ciliated cells. Therefore the local and systemicanti-B. pertussis antibody responses in BPAL10-immunized infant micewere measured two months after a single i.n. administration of 5×10³ CFUand compared to the antibody responses measured in BPZE1-immunizedage-matched animals. BPAL10-immunized mice produced comparable levels ofB. pertussis-specific IgA (FIG. 3A) and slightly higher levels of B.pertussis-specific IgG antibodies (FIG. 3B) in their BALFs compared toBPZE1-immunized mice. The levels of systemic anti-B. pertussis IgG werecomparable between the two groups (FIG. 3D), whereas no significantserum IgA response was detected (FIG. 3C).

Mice were i.n. administered with 5×10³ CFU of BPZE1 or BPAL10. Sera werecollected 2 months post-infection and analyzed by ELISA for anti-B.pertussis IgG1, IgG2a, IgG2b and IgG3. Sera from individual mice ofnaive group and BPZE1 were pooled, while serum from eachBPAL10-immunized mouse was tested individually. Sera from all groupswere diluted 1/50 prior to testing. The data presented is representativeof two independent experiments. Analysis of the anti-B. pertussis IgGsubtype indicates that BPAL10-immunized mice produced comparable levelsof IgG2a and IgG2b antibodies to those measured in BPZE1-immunized mice,suggestive of a Th1-biased immune response triggered in both animalgroups, and demonstrating that the absence of CyaA in BPAL10 did notaffect the anti-B. pertussis IgG isotype profile (Table 1).

TABLE 1 Anti-B. pertussis IgG isotyping in immune sera. Immunogen Mouseserum Absorbance at 490 nm of serum at 1/50 dilution IgG2aa/ IgG1 IgG2aIgG2b IgG3 IgG1 Naïve Pooled 0.15 0.08 0.09 0.14 0.57 BPZE1 Pooled 1.212.68 2.96 1.27 2.22 BPAL10 M1 0.87 2.85 2.95 1.66 3.30 M2 1.68 2.89 2.952.15 1.72 M3 1.78 2.50 2.92 2.24 1.40 M4 1.79 2.81 2.99 1.63 1.57 M51.19 2.67 3.14 1.33 2.25 M6 1.13 2.92 2.96 0.98 2.58

IFN-γ, IL-4 and IL-17 production: Studies in mice have demonstrated thatprotection is maintained after waning of circulating antibodies, throughthe generation of immunological memory. IFNγ plays an essential role incontrolling B. pertussis infection, and disruption of the gene codingfor the IFNγ receptor has been shown to result in an atypicaldisseminated lethal disease characterized by pyogranulomatousinflammation and post necrotic scarring in the livers, mesenteric lymphnodes and kidneys. Therefore, to assess the IFNγ response ofBPAL10-immunized infant mice, their spleens were harvested 2 monthspost-infection and the splenocytes were re-stimulated in vitro with B.pertussis whole cell lysate. Significantly lower levels of IFNγ weremeasured in the culture supernatant of the re-stimulated BPAL10-primedsplenocytes compared to the levels measured in the supernatant ofBPZE1-primed splenocytes, with no significant difference in IL-4 andIL-17 (FIG. 4A-C). Whether the reduced IFNγ production by BPAL10-primedsplenocytes could be attributed to this cell population wasinvestigated. However, comparable percentages of IFNγ-producing CD4+ Tcells were observed in both BPAL10- and BPZE1-primed splenocytes (FIG.4D) thereby suggesting that the reduced IFNγ production observed withBPAL10-primed splenocytes cannot be attributed to a defective activationof CD4+ T cells.

Protective efficacy against pertussis challenge: To assess the abilityof BPAL10 to protect against subsequent B. pertussis infection, 3week-old BALB/c mice were i.n. infected with either 5×10⁵ or 5×10³ CFUof BPZE1 or BPAL10, and challenged with virulent B. pertussis BPSM onemonth later. The results indicate that at 5×10⁵ CFU, a single i.n.immunization with BPAL10 protects as efficiently as BPZE1, as evidencedby a comparable bacterial clearance between the two groups (FIG. 5A).However, with the 5×10³ CFU vaccine dose, the BPSM bacterial loads weresignificantly higher in the BPAL10-immunized mice compared to miceimmunized with BPZE1. Thus, these results support that BPZE1 offers amore robust protection to infant mice than its CyaA-deficientcounterpart BPAL10 (FIG. 5B).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of immunizing a mammalian subject toprotect it from developing pertussis, the method comprising the step ofcolonizing the respiratory tract of a mammalian subject with a vaccinecomprising a sufficient amount of a live attenuated Bordetella pertussisstrain that has been rendered deficient in functional adenylate cyclasetoxin (CyaA), functional pertussis toxin (PTX), functional dermonecrotictoxin (DNT), and functional tracheal cytotoxin (TCT) to elicit an immuneresponse that prevents the mammalian subject from developing pertussis.2. The method of claim 1, wherein the strain comprises a gene encodingCyaA which has been mutated such that the strain fails to produce CyaA.3. The method of claim 1, wherein the strain comprises a gene encodingPTX which has been mutated such that the strain fails to producefunctional PTX.
 4. The method of claim 1, wherein the strain lacks agene encoding functional DNT.
 5. The method of claim 1, wherein thestrain lacks a functional wild-type ampG gene.
 6. The method of claim 5,wherein the strain comprises a heterologous ampG gene.
 7. The method ofclaim 1, wherein the strain comprises: a gene encoding CyaA which hasbeen mutated such that the strain fails to produce functional CyaA and agene encoding PTX which has been mutated such that the strain fails toproduce functional PTX; and lacks a gene encoding functional DNT, and afunctional wild-type ampG gene.
 8. The method of claim 1, wherein thestrain is BPAL10.
 9. The method of claim 1, wherein the vaccine isprovided in a single dosage form which comprises at least 5×10⁵ colonyforming units (CFU) of the strain.